Systematic analysis of Heat Shock Protein 70 (HSP70) gene family in radish and potential roles in stress tolerance

The 70 kD heat shock proteins (HSP70s) represent a class of molecular chaperones that are widely distributed in all kingdoms of life, which play important biological roles in plant growth, development, and stress resistance. However, this family has not been systematically characterized in radish (Raphanus sativus L.). In this study, we identified 34 RsHSP70 genes unevenly distributed within nine chromosomes of R. sativus. Phylogenetic and multiple sequence alignment analyses classified the RsHSP70 proteins into six distinct groups (Group A–F). The characteristics of gene structures, motif distributions, and corresponding cellular compartments were more similar in closely linked groups. Duplication analysis revealed that segmental duplication was the major driving force for the expansion of RsHSP70s in radish, particularly in Group C. Synteny analysis identified eight paralogs (Rs-Rs) in the radish genome and 19 orthologs (Rs-At) between radish and Arabidopsis, and 23 orthologs (Rs-Br) between radish and Chinese cabbage. RNA-seq analysis showed that the expression change of some RsHSP70s were related to responses to heat, drought, cadmium, chilling, and salt stresses and Plasmodiophora brassicae infection, and the expression patterns of these RsHSP70s were significantly different among 14 tissues. Furthermore, we targeted a candidate gene, RsHSP70–23, the product of which is localized in the cytoplasm and involved in the responses to certain abiotic stresses and P. brassicae infection. These findings provide a reference for further molecular studies to improve yield and stress tolerance of radish. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04653-6.


Background
Heat shock proteins (HSPs) are conserved stress-responsive proteins induced by adverse environmental conditions, which can be divided into six major subfamilies according to their molecular masses, i.e., HSP40, HSP60, HSP70, HSP90, HSP100, and small HSPs (sHSPs) [1].HSP70 is the most widely studied type of HSP and is characterized by the N-terminal ATPase domain (NBD), substrate binding domain (SBD), and a variable C-terminal lid region domain [2].Based on the subcellular localization, HSP70s in plants have been classified into four major subfamilies: those present in the cell nucleus/ cytoplasm (EEVD motif ), endoplasmic reticulum (HDEL motif ), plastids (PEGDVIDADFTDSK motif ), and mitochondria (PEAEYEEAKK motif ) [3].
As molecular chaperones, the most important biological function of HSP70s is linked to acquired thermotolerance under high-temperature stress, and function as negative feedback regulators of heat shock transcription factor (HSF) activity [4][5][6][7].In Arabidopsis, cpHsc70-1(At4g24280) is not only essential for normal plant growth but is also important for root growth from heat-stressed seeds [8].AtHSP70-15 affects not only the growth phenotype and leaf morphology but also the response to heat stress [9].Mutation of the rice chloroplast OsHsp70CP1 causes chloroplast developmental defects under high-temperature conditions [10].Plastid HSP70-2 (cpHsp70-2) involved with the temperaturedependent chalkiness of rice grains [11].HSP70s have also been studied under high temperature stress in a variety of vegetables, such as pepper [12], pumpkin [13], potato [14], cucumber [15] and tomato [16].In addition, HSP70 proteins in plants also improve tolerance to lowtemperature, high-salinity, drought, light, flooding, and heavy metal stress in Arabidopsis, maize, rice, barley, wheat, soybean, tobacco, poplar, sugarcane, and chlamydomonas [16].Furthermore, some HSP70s in plants are also involved in microbial pathogenesis, particularly viral infections [17].Cytoplasmic HSP70s enhance the infection of Nicotiana benthamiana by tobacco mosaic virus, potato virus x, cucumber mosaic virus, and watermelon mosaic virus [18].HSP70-depleted plants show increased susceptibility to Pseudomonas syringae [19], and silencing of cytosolic HSP70a in pepper results in enhanced susceptibility to Xanthomonas campestris infection [20].Plants show enhanced resistance to infection by rice stripe virus (RSV) [21] through the accumulation of endoplasmic reticulum (ER)-resident HSP70s (also called binding proteins, BiPs).
Radish (Raphanus sativus L., 2n = 18) is an important commercial root vegetable crop that belongs to the Brassicaceae family and is sensitive to heat [22], salt [23] or heavy metal stress [24][25][26].Although the HSP70 gene family is associated with different abiotic stress responses in many plant species, the genome-wide identification and functional characterization of the HSP70 family in radish have not been reported previously.In this study, 34 HSP70 family members were identified in the radish genome and divided into different classes, and the phylogenetic relationships, chromosome arrangements, gene structures, conserved motifs, and expression profiles of RsHSP70 genes in distinct tissues or in response to various environmental stresses were also systematically analyzed.Our results provide a biological reference for elucidating the functions of HSP70 genes in radish, and will be useful for the selection of candidate genes for genetic engineering in R. sativus breeding.

Expression analysis of RsHSP70 genes in various tissues or under different stresses
To examine the potential functions of RsHSP70 genes in different tissues of radish, the raw RNA-seq data of 14 tissues were acquired from a previous report [42] (Table S 1).These data represented five tissues (leaf, root tip, cortex, cambium, and xylem) and five stages (7,14,20,40, and 60 days after sowing, DAS).To investigate whether RsHSP70 genes play important roles in various stress responses in radish, data from seedlings exposed to heat, drought, cadmium (Cd), chilling, salt stress were obtained from the NCBI SRA database (SUB13505845) and P.brassicae infection were obtained from the CNCB database (https:// ngdc.cncb.ac.cn/) with the number CRA004024.The expression level of each gene presented in transcripts fragments per kilobase of exon model per million mapped fragments (FPKM) was reanalyzed as described previously [43].Differentially expressed genes (DEGs) were selected with cutoff values of |log2fold-change|> 1 and p < 0.05.Heat maps were generated in R (v3.6.3) with log2-transformed FPKM values after the addition of a pseudocount of 0.01.

Plant material and treatment
Two inbred radish lines with contrasting resistance to P. brassicae, WR1150 and 1116 T, (resistant and susceptible, respectively) obtained from the Institute of Vegetables and Flowers, Chongqing Academy of Agricultural Sciences, China, were used.The seeds of the two lines were surface-sterilized, sown in trays containing a 3:1 mixture of nutrient soil and sand, and grown under conditions of 25 °C/20 °C (day/night) with a 16 h photoperiod.The pathogen used in this study was obtained from the natural clubroot nursery at Wulong, Chongqing, China, where P. brassicae race 4 is the dominant clubroot pathogen.P. brassicae suspensions were prepared as described previously [44], and inoculate we prepared by diluting the suspension to a resting spore concentration of 1 × 10 8 /mL.Ten similar seedlings with 20-day leaves were individually injected at the bottom of the stem with 5 mL spore suspension.Total RNA was isolated from roots collected at 0, 4, 7, 14, 21, and 28 days post inoculation (dpi) of P. brassicae for qRT-PCR analysis.

RNA isolation and qRT-PCR analysis
Total RNA was isolated from 20-day leaves using an RNAprep pure Plant Kit (Catalog no.dp432; Tiangen Biotech Co., Ltd., Beijing, China), and quantified using the NanoDrop ND-1000 (Termo Scientifc, Waltham, MA, USA).The first-stand cDNA was synthesized using a HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Catalog no.R312-01; Vazyme Biotech Co., Ltd., Nanjing, China).ChamQ Universal SYBR qPCR Master Mix (Catalog no.Q711-02; Vazyme Biotech) was used for qRT-PCR on a BIO-RADCFX96 Real Time System (Bio-Rad Laboratories, Hercules, CA, USA).Three independent biological replicates were used for each sample.The transcript levels were calculated using the ΔΔC T method with normalization relative to the level of RsActin [45].All gene-specific primers used in this study are presented in Table S 2.

Yeast constructs, tolerance assay and growth curve
The coding sequence of RsHSP70-23 was inserted into pRS-416-GFP, and the recombinant vector and the empty pRS-416-GFP vector (control) were transformed into the wild-type strain JRY472 and allowed to grow on SD-Ura plates.Positive recombinant transformants cultured in SD-Ura medium were diluted until OD 600 = 0.1.Then the cell culture was diluted ten fold and treated with 75 µM Cd, 1 M NaCl, and 2 M mannitol, and incubated at 30 °C for 3 days.Cold and heat stress were applied at 4 °C and 37 °C for 2 days before transfer to 30 °C for 1 or more days [46].Then, the phenotypes of the yeast cells were photographed, and the experiment was repeated three times.RsHSP70-23 overexpressing yeast cells were grown in liquid SD-Ura medium at 4℃, 30℃ and 37℃, respectively, and with 1 M NaCl were also grown at 30℃, and were diluted until OD600 = 0.1, OD600 is recorded every 2 h to prepare cell growth curve [46].

Identification of RsHSP70 genes in radish genome
Through a combination of BLASTP and HMM profile analysis, 34 putative RsHSP70 genes were identified in the "Xiangyabai" genome and renamed according to their relative linear order on each chromosome (Table 1).Among them, the lengths of predicted RsHSP70 protein sequences varied from 112 (RsHSP70-32) to 1335 (RsHSP70-4) amino acids with molecular weights (MWs) ranging from 12.21 to 144.26 kDa.The theoretical isoelectric points (pIs) of most predicted RsHSP70 proteins were < 7, with the exceptions of RsHSP70-4, RsHSP70-29, RsHSP70-30, and RsHSP70-32.In addition, WoLF PSORT online analysis predicted that within the 34 RsHSP70 proteins, 19 radish HSP70 proteins were localized to the cytosol/nucleus, 7 to the chloroplast, 4 to the mitochondria, and 4 to the ER (Table 1).

Six groups defined among the HSP70 genes of six species
To investigate the phylogenetic relationships of these HSP70s, the full-length amino acid sequences of RsH-SP70s (34), BnHSP70s (Brassica napus, 47), BrHSP70s (Brassica rapa, 29), BoHSP70s (Brassica oleracea, 20), OsHSP70s (Oryza sativa, 32), and AtHSP70s (Arabidopsis thaliana, 18) were used to construct a neighborjoining (NJ) phylogenetic tree; they clustered into six distinct groups (Groups A-F) (Fig. 1).Among them, Group A was the most abundant subfamily containing 58 members, consisting of 13 RsHSP70 members that were predicted to be localized in the cytoplasm/ nucleus (Table 1).Groups B, C, and D consisted of four, five, and four members from radish, which were predicted to be located in the ER, chloroplast, and mitochondria, respectively.These results suggest that the closely related HSP70s are usually located in the same subcellular structure.In total, 38 members belonging to the Hsp110/SSE subfamily were classified into Group F, which also contained 7 RsHSP70 members.Group E only had one RsHSP70, which was suggested to be a truncated gene based on comparative analysis with its Arabidopsis counterpart [47].Each group contained proteins from both monocotyledonous and dicotyledonous plants, indicating that the main characteristics of HSP70 proteins in plants emerged before the separation of these two lineages.

Characterization of RsHSP70 proteins and distributions of conserved motifs and gene structures
Sequence alignments revealed that most HSP70 family proteins in radish included three distinct domains.The highly conserved NBD domain possessed three HSP70 signature sequences, the SBD domain was also conserved, while the C-terminal domain was highly variable (Figure S1).However, RsHSP70-14 and RsHSP70-27 did not include the NBD domain, and RsHSP70-32 and RsHSP70-33 lacked the C-terminal domain.All 34 RsHSP70s clustered into six groups in phylogenetic analysis (Fig. 2A).The majority (11/13, 84.6%) of RsHSP70s in Group A possessed a conserved retention signal "EEVD" sequence at the C-terminus, and were localized in the cytoplasm.Half (2/4) of those in Group B were localized in the ER and 60% (3/5) of those in Group C were localized in the chloroplasts, and possessed the conserved C-terminal signature sequences "HDEL" and "DVIDADFTDSK, " respectively.In addition, the three RsHSP70s in Group D possessed the conserved signature sequence "PEAEYEEAKK" in the C-terminus, and were localized in the mitochondria (Figure S1).
Twenty conserved motifs were identified in radish HSP70 proteins using the MEME motif search tool (Fig. 2B and Table S 3).Motifs 2 (24/34), 3 (26/34), and 12/10 (22/34) were found in the RsHSP70 family, corresponding to signature sequences.Motifs 1 (23/34), 5 (29/34), and 7 (25/34) were included in the SBD domain.Similar compositions, orders, and numbers of motifs were found in the groups localized to the mitochondria and chloroplasts.However, some subfamilies also included several specific motifs.For example, motifs 13 and 14 were exclusively present in the HSP110/SSE subfamily, which lacked motif 1; motif 19 was only found in those localized to the cytosol; and motifs 16 and 20 were absent in those localized to the cytosol or ER.
The online GSDS tool was used to identify the exonintron organization in the coding sequences shared among the RsHSP70s (Fig. 2C).The number of exons in RsHSP70s varied from 1 to 14.Most closely related members that clustered together shared similar exon-intron organization and exon length.For example, the two members of the HSP110/SSE subfamily have 13 introns and 14 exons, and are nearly 2700 bp in length, while 84.6% of cytosolic HSP70s had no introns.These results indicate a diversity of exon-intron organization in the radish HSP70 family.

Gene duplication and synteny analysis of RsHSP70s
Chromosome location analysis showed that in addition to RsHSP70-34 the remaining 33 RsHSP70 genes were irregularly distributed on the nine chromosomes in radish (Fig. 3).Chromosome 8 had the greatest number of HSP70 genes (n = 6), followed by five genes on each of chromosomes 2 and 6, four genes on chromosomes 4, three genes on each of chromosomes 1, 7 and 8, and only two on each of chromosomes 3 and 9.

Potential roles of HSP70 genes in radish root with P. brassicae infection
Most HSP70 genes in Arabidopsis are upregulated in response to biotic stressors [17].To explore the roles of RsHSP70s genes responsive to biotic stress, we compared the expression profiles of all RsHSP70 genes in the roots at 0, 7, and 28 dpi with P. brassicae (Fig. 7).In all, 11 RsHSP70 genes were either up-or downregulated and showed significant differential expression in response to P. brassicae infection between the clubroot-resistant (RB) and clubroot-susceptible (SB) lines.Only RsHSP70-5, RsHSP70-23, and RsHSP70-33 tended to show continuous upregulation at all time points examined.It is noteworthy that the level of RsHSP70-23 expression was 6.83-fold higher in RB than SB roots at 28 dpi (Fig. 7).qRT-PCR analysis showed that the level of RsHSP70-23 transcript increased sharply in the clubroot-resistant strain (WR1150) compared to the clubroot-susceptible strain (1116 T) after 4 dpi, and remained at a high level from 7 to 28 dpi.These results suggest that RsHSP70-23 may participate in the response to P. brassicae infection in radish.

Subcellular localization of RsHSP70-23
Large-scale synteny analysis revealed that RsHSP70-23 was orthologous to AT5G42020 (BIP2), which is localized to the ER (Fig. 4).WoLF PSORT analysis showed that RsHSP70-23 was also localized to the ER (Table 1), but did not possess the conserved "HDEL" sequence in the C-terminus (Figure S1).To determine the actual subcellular localization of RsHSP70-23 in vivo, GFP-tagged RsHSP70-23 was transiently expressed in Arabidopsis protoplasts.Free GFP was evenly distributed in the cytoplasm as expected, and RsHSP70-23-GFP fusion protein was also observed in the cytoplasm (Fig. 8).

RsHSP70-23 overexpression in response to abiotic stresses in yeast
To examine whether expression of RsHSP70-23 responds to abiotic stress, RsHSP70-23-overexpressing yeast cells were exposed to 75 µM Cd, 1 M NaCl, 2 M mannitol, chilling (4 °C), and heat (37 °C) stresses (Fig. 9).There were no differences between cells carrying the RsHSP70-23 overexpression vector or empty vector (EV) control under conditions of mannitol or Cd stress.However, the RsHSP70-23-overexpressing cells were sensitive to cold stress compared to EV.Under conditions of NaCl and heat stress, all cells overexpressing RsHSP70-23 grew faster than those carrying EV.In addition, we conducted growth curves of the yeast cells under heat, chilling and Fig. 8 The subcellular localization of RsHSP70-23.Bars = 10 μm salt stresses (Fig. 10).The growth rate of the RsHSP70-23-overexpressing cells showed no difference compared with that of EV under normal conditions, and under salt and heat stress, the optical density was higher than that of EV, whereas under chilling stress, the optical density was decreased compared with that of EV.These results indicated that overexpression of RsHSP70-23 can alleviate the adverse effects of high temperature and salinity stress on yeast cell growth.

Discussion
HSP70 proteins have been characterized as molecular chaperones expressed in a variety of plants, and most HSP70 family genes play important roles in the regulation of plant growth, development, and defense [16].In the present study, 34 potential HSP70 genes were identified based on the R. sativus genome sequence, and were divided into six major subfamilies (Fig. 1 and Table 1).Within the same subfamilies, the most closely related RsHSP70 members shared more similarity in terms of motif composition, exon-intron organization, and the corresponding cellular compartments (Fig. 2).Notably, the number of HSP70 members varied among the species tested regardless of genome size and chromosome number (Fig. 1).Radish has twice as many HSP70s as Arabidopsis, which is inconsistent with a whole genome triplication (WGT) event that occurred in the Brassicaceae family [48], indicating that some RsHSP70 genes were lost after expansion.Segmental duplication was the main mechanism of RsHSP70 gene expansion, accounting for 32.35% of cases (Fig. 3).Segmental duplication occurred 2.49-7.58Mya (Table 2), indicating that the duplication event of all pairs occurred after the divergence of Arabidopsis and Brassica at about 14.5-20.4Mya [48], and also after divergence from cabbage and Chinese cabbage at approximately 7.1-10.4Mya [49].In addition, approximately 57.15% of segmentally duplicated genes had a Ka/Ks value < 0.1 (Table 2), indicating strong purifying selection, and these gene pairs may have become conserved and their functions tended to be constrained [31].
After duplication, genes may be maintained through subfunctionalization and/or neofunctionalization at the expression or sequence level.Alternatively, duplicated copies may accumulate deleterious mutations and become nonfunctional pseudogenes [50].In this study, we found some sister pairs with changes in their exonintron structures and numbers; RsHSP70-27 contained 2 exons, while its paralogs RsHSP70-4 and RsHSP70-11 had 11 and 4 exons, respectively (Fig. 2), indicating that exons were lost during evolution, similar to reports in potato and soybean [12,51].Large-scale synteny analysis showed that AT4G24280 had three orthologous genes in radish (RsHSP70-4, RsHSP70-11, and RsHSP70-27) (Fig. 4).RsHSP70-27 was not expressed in any of the tissues and/or stress conditions examined, suggesting that it is undergoing pseudogenization.However, RsHSP70-4 and RsHSP70-11 exhibited distinct expression patterns between both tissues and various abiotic stress conditions, indicating neofunctionalization (Figs. 5 and 6 and Tables S 5 and S 6).Moreover, there were 13 common syntenic genes between radish, Chinese cabbage, and Arabidopsis, thus providing a valuable reference for further understanding of the biological functions of these homologous genes in radish.
HSP70 genes are key components in plant development and in responses to a wide range of abiotic stresses [16].We found that most RsHSP70s exhibited diverse expression profiles in different tissues, particularly in the 20-day leaves and 20-day roots of radish plants (Fig. 5 and Table S 5), suggesting that HSP70 family genes may play important roles in radish seedling development [47].Under conditions of abiotic stress, transcription factors bind to the cis-regulatory elements of stress-responsive gene promoters and specifically initiate transcription of the corresponding genes [51].In potato, most StHSP70s respond to various abiotic stresses (salt, drought, heat, and cold) and hormone treatments (ABA, IAA, GA3, and SA) [12].More than half of the HSP70 genes are responsive to ABA, drought, and salt stresses in rice, Arabidopsis, and moss [52].ABA (ABRE), MeJA (CGTCA-motif and TGACGmotif ), SA (TCA element), drought (MBS), cold (LTR), and heat (HSE) response elements have been observed in the promoter regions of PyyHSP70 genes [53].Here, a variety of hormone and stress response elements were found in the promoter regions of RsHSP70 family genes (Table S 7), where the numbers of HSEs were significantly greater than those of other elements (Table S 8).The two main subunits (5′NGAAN3′ and 5′NTTCN3′) of HSE are recognized by HSF1 [54].As molecular chaperones, the most important biological function of HSP70s is related to acquired thermotolerance under heat stress, and their expression functions as a negative feedback regulator of heat shock transcription factor (HSF) activity [4][5][6][7].Among the five stress conditions examined here, heat stress induced the greatest number of HSP70 genes in radish (Fig. 6A).These findings suggest that the RsHSP70 genes may respond to multiple hormones and abiotic stresses, particularly heat stress, in radish plants.
Cytosolic and ER-resident HSP70s also play essential regulatory roles in the innate immune response in plant cells [17].AtBIP2 is localized to the ER and upregulated in response to Sclerotinia sclerotiorum, P. syringae, and FLG-22.In phylogenetic analysis, RsHSP70-23 clustered with AtBIP2 of Group B (Fig. 1), and large-scale synteny analysis also showed that RsHSP70-23 was orthologous with AtBIP2 (Fig. 4 and Table S 4).The expression levels of RsHSP70-23 in response to P. brassicae infection were significantly higher in clubroot-resistant than clubroot-susceptible lines (Fig. 7).In addition, BiP genes are induced by multiple abiotic stressors [17,52].We found that RsHSP70-23 showed a lower rate of expression in all tissues examined (Fig. 5) but was significantly upregulated under conditions of salt and heat stress (Figs. 6, 9 and 10).Surprisingly, analysis of the subcellular localization of RsHSP70-23-GFP fusion protein indicated exclusive cytoplasmic localization in Arabidopsis protoplasts (Fig. 8).These results suggest that the functions Fig. 10 Growth curves of the RsHSP70-23 gene overexpressing yeast cells and EV (empty vector (yeast WT)) under normal, heat, salt and chilling stress.Cell density was monitored after 12, 14, 16, 18, 20, 22 and 24 h after the treatment.The error bar represents the deviation of three independent replications of RsHSP70-23 proteins may have been conserved after the divergence of radish and Arabidopsis but also exhibit unique functions through changes in localization during adaptation to changes in the environment.

Conclusions
In summary, 34 RsHSP70 genes were identified in the radish genome.Their physiochemical properties, phylogenetic relationships, gene organization, gene structures, chromosome distribution, and gene duplication were analyzed, and their expression patterns were characterized to understand their critical functions.These genes may play crucial roles in the growth, development, and stress responses of radish.In addition, RsHSP70-23 was localized to the cytoplasm and was involved in responses to certain abiotic stressors and P. brassicae infection.This comprehensive characterization of the RsHSP70 gene family will facilitate analysis of HSP70-gene mediated molecular mechanisms of stress responses in root vegetable crops.

Fig. 1 Fig. 2
Fig. 1 Phylogenetic relationships of radish, rapeseed, Chinese cabbage, cabbage, rice, and Arabidopsis HSP70 proteins.The tree was divided into six subgroups, marked by different color backgrounds

Fig. 3
Fig. 3 Chromosomal localization and gene duplication events of RsHSP70 genes.Respective chromosome numbers are indicated at the top of each bar.Tandem duplicated genes are marked on a grey background.Segmental duplicated genes are shown by black line

Fig. 5
Fig. 5 Expression profiles of RsHSP70 genes in different tissues.A The normalized expression levels of the hierarchical clustering of 34 RsHSP70 genes in 14 tissues.The relative expression levels corresponding to the log2-transformed TPM values after the addition of a pseudocount of 0.01 are shown.The scale represents the relative signal intensity of the TPM values.B The expression values of all the RsHSP70 genes in each tissue

Fig. 7
Fig. 7 Expression profiles of RsHSP70 genes in response to P.brassicae infection.A Heatmap of differential expression of RsHSP70 genes in response to P.brassicae infection in RB and SB at 0, 7 and 28 dpi.The color scale of heatmap is based on the log2Foldchange values between RB and SB are shown.B RT-PCR analysis of selected genes under P. brassicae infection in WR1150 and 1116 T at 0, 4, 7, 14, 21 and 28 dpi.Error bars indicate the SD for three independent replicates.* and ** indicate a signifcant diference between WR1150 and 1116 T at P < 0.05 and P < 0.01 levels, respectively (two tailed T-test)

Table 2
Ka-Ks calculation for each pair of HSP70 in radish S-sitesNumber of synonymous sites, N-sites Number of non-synonymous sites, Ka Non-synonymous substitution rate, Ks Synonymous substitution, Mya Million years ago